PROJECT SUMMARY We have shown that some types of engineered nanomaterials (ENMs) induce oxidative stress, alter macrophage gene regulation and phagocytic function, and increase the susceptibility of mice to Streptococcus pneumonia, the leading cause of community-acquired pneumonia. Increased risks of pneumonia are a major health outcome associated with human exposure to ultrafine environmental particulates, particularly in the elderly. Our goal is to develop and test a framework for prediction of ENM effects on infection susceptibility, using mechanism-based in vitro assays of macrophage function. We hypothesize exposure to ENMs compromises innate immune function and enhances susceptibility to lung infections through redox-mediated signaling mechanisms that alter macrophage polarization and impair phagocytic clearance of pathogens. We also propose that protein S-glutathionylation (SSG), a major form of protein oxidative modification that regulates multiple aspects of innate immunity, contribute as molecular initiative events for ENM toxicity. Aim 1 will use novel redox protoemics methods to identify the protein targets of SSG induced by ENMs and the major pathways that are most sensitive to these modifications. Aim 2 will identify macrophage transcriptional pathways impacted by ENMs and how exposure to ENMs modulate phagocytic activity toward S. pneumonia. We will investigate how the most robust SSG modifications and mRNA pathways affected by ENMs are co- regulated, and develop sensitive assays to quantify these markers in vitro and in vivo. Aim 3 will test whether in vitro assays for macrophage function and pathway markers identified in Aims 1-2 accurately predict whether inhalation exposure to ENMs modulates lung infections in both young and aged mice challenged with S. pneumonia. Advanced nanomaterial dosimetry models will be used to derive equivalent human exposure levels that would be required to induce these effects. The ability to accurately predict adverse outcomes of ENMs from mechanism-based in vitro studies will transform hazard and risk approaches for emerging ENMs, and addresses a broader human health problem associated with environmental particulate exposures.